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The Spanish Society for Developmental Biology organized its 17th meeting in November 2020. The meeting, organized by CIC bioGUNE, the University of the Basque Country and the University of Cantabria, gathered about 280 registrants and received 132 scientific abstracts. Participants ranged from undergraduate to senior researchers, with a broad participation of Ph.D. students. The meeting was organized in 8 sessions: Growth and Scaling, Self-organization, Neurodevelopment, Genomes, Cell Biology, Development and Disease, Evo-Devo and Regeneration (Araújo et al., 2021). These sessions focused on the new tendencies in Developmental Biology research and, based on the science presented there, we organized this special issue on The 17th Edition of the Spanish Society for Developmental Biology Meeting: New Trends in Developmental Biology. This collection of articles gathers several scientific contributions in this area, featuring collaborative and interdisciplinary approaches among developmental biologists. With the focus on organogenesis and gene regulation, our selected content embraces novel discoveries on muscle development, regeneration and the transcriptional control and role of miRNAs in development, while highlighting advances in organogenesis and gonadal development.

The Spanish Society for Developmental Biology (SEBD) organized its 17th meeting in November 2020 (herein referred to as SEBD2020). This meeting, originally programmed to take place in the city of Bilbao, was forced onto an online format due to the SARS-CoV2, COVID-19 pandemic. Although, we missed the live personal interactions and missed out on the Bilbao social scene, we were able to meet online to present our work and discuss our latest results. An overview of the activities that took place around the meeting, the different scientific sessions and the speakers involved are presented here. The pros and cons of virtual meetings are discussed.

Branching networks are a very common feature of multicellular animals and underlie the formation and function of numerous organs including the nervous system, the respiratory system, the vasculature and many internal glands. These networks range from subcellular structures such as dendritic trees to large multicellular tissues such as the lungs. The production of branched structures by single cells, so called subcellular branching, which has been better described in neurons and in cells of the respiratory and vascular systems, involves complex cytoskeletal remodelling events. In Drosophila, tracheal system terminal cells (TCs) and nervous system dendritic arborisation (da) neurons are good model systems for these subcellular branching processes. During development, the generation of subcellular branches by single-cells is characterized by extensive remodelling of the microtubule (MT) network and actin cytoskeleton, followed by vesicular transport and membrane dynamics. In this review, we describe the current knowledge on cytoskeletal regulation of subcellular branching, based on the terminal cells of the Drosophila tracheal system, but drawing parallels with dendritic branching and vertebrate vascular subcellular branching.

SUMMARYThe establishment of branched structures by single cells involves complex cytoskeletal remodelling events. InDrosophila, epithelial tracheal system terminal cells (TCs) and dendritic arborisation neurons are models for these subcellular branching processes. During tracheal embryonic development, the generation of subcellular branches is characterized by extensive remodelling of the microtubule (MT) network and actin cytoskeleton, followed by vesicular transport and membrane dynamics. We have previously shown that centrosomes are key players in the initiation of subcellular lumen formation where they act as microtubule organizing centres (MTOCs). However, not much is known on the events that lead to the growth of these subcellular luminal branches or what makes them progress through a particular trajectory within the cytoplasm of the TC. Here, we have identified that the spectraplakinShort-stop(Shot) promotes the crosstalk between MTs and actin, which leads to the extension and guidance of the subcellular lumen within the TC cytoplasm. Shot is enriched in cells undergoing the initial steps of subcellular branching as a direct response to FGF signalling. An excess of Shot induces ectopic acentrosomal branching points in the embryonic and larval tracheal TC leading to cells with extra subcellular lumina. These data provide the first evidence for a role for spectraplakins in subcellular lumen formation and branching.

The specification of tissue identity during embryonic development requires precise spatio-temporal coordination of gene expression. Many transcription factors required for the development of organs have been identified and their expression patterns are known; however, the mechanisms through which they coordinate gene expression in time remain poorly understood. Here, we show that hormone-induced transcription factor Blimp-1 participates in the temporal coordination of tubulogenesis in Drosophila melanogaster by regulating the expression of many genes involved in tube maturation. In particular, we demonstrate that Blimp-1 regulates the expression of genes involved in chitin deposition and F-actin organization. We show that Blimp-1 is involved in the temporal control of lumen maturation by regulating the beginning of chitin deposition. We also report that Blimp-1 represses a variety of genes involved in tracheal maturation. Finally, we reveal that the kinase Btk29A serves as a link between Blimp-1 transcriptional repression and apical extracellular matrix organization., S.J.A. was a Ramon y Cajal Researcher (RYC-2007-00417); A.O. was the recipient of an IRB-La Caixa fellowship. This work was supported by grants from the Generalitat de Catalunya and the Spanish Ministerio de Ciencia e Innovación (BFU2009-07629).

Through complex mechanisms that guide axons to the appropriate routes towards their targets, axonal growth and guidance lead to neuronal system formation. These mechanisms establish the synaptic circuitry necessary for the optimal performance of the nervous system in all organisms. Damage to these networks can be repaired by neuroregenerative processes which in turn can re-establish synapses between injured axons and postsynaptic terminals. Both axonal growth and guidance and the neuroregenerative response rely on correct axonal growth and growth cone responses to guidance cues as well as correct synapses with appropriate targets. With this in mind, parallels can be drawn between axonal regeneration and processes occurring during embryonic nervous system development. However, when studying parallels between axonal development and regeneration many questions still arise; mainly, how do axons grow and synapse with their targets and how do they repair their membranes, grow and orchestrate regenerative responses after injury. Major players in the cellular and molecular processes that lead to growth cone development and movement during embryonic development are the Soluble N-ethylamaleimide Sensitive Factor (NSF) Attachment Protein Receptor (SNARE) proteins, which have been shown to be involved in axonal growth and guidance. Their involvement in axonal growth, guidance and neuroregeneration is of foremost importance, due to their roles in vesicle and membrane trafficking events. Here, we review the recent literature on the involvement of SNARE proteins in axonal growth and guidance during embryonic development and neuroregeneration, Research in our laboratories was supported by the Ramon y Cajal programme (RYC-2007-00417, RYC-2009-05510) and grants from the Spanish MINECO (SAF2013-42445R and BFU2010-21507) and CIBERNED.

Axonal growth and guidance rely on correct growth cone responses to guidance cues, both in the central nervous system (CNS) and in the periphery. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the cross-talk mechanisms between guidance and membrane dynamics and turnover in the axon. Our studies have shown that Netrin-1/deleted in colorectal cancer signaling triggers exocytosis through the SNARE Syntaxin-1 (STX-1) during the formation of commissural pathways. However, limited in vivo evidence is available about the role of SNARE proteins in motor axonal guidance. Here we show that loss-of-function of SNARE complex members results in motor axon guidance defects in fly and chick embryos. Knock-down of Syntaxin-1, VAMP-2, and SNAP-25 leads to abnormalities in the motor axon routes out of the CNS. Our data point to an evolutionarily conserved role of the SNARE complex proteins in motor axon guidance, thereby pinpointing an important function of SNARE proteins in axonal navigation in vivo. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 963-974, 2017.

The centrosome, a major microtubule organizer, has important functions in regulating the cytoskeleton as well as the position of cellular structures and orientation of cells within tissues. The centrosome serves as the main cytoskeleton-organizing centre in the cell and is the classical site of microtubule nucleation and anchoring. For these reasons, centrosomes play a very important role in morphogenesis, not just in the early stages of cell divisions but also in the later stages of organogenesis. Many organs such as lung, kidney and blood vessels develop from epithelial tubes that branch into complex networks. Cells in the nervous system also form highly branched structures in order to build complex neuronal networks. During branching morphogenesis, cells have to rearrange within tissues though multicellular branching or through subcellular branching, also known as single-cell branching. For highly branched structures to be formed during embryonic development, the cytoskeleton needs to be extensively remodelled. The centrosome has been shown to play an important role during these events.

The centrosome, a major microtubule organizer, has important functions in regulating the cytoskeleton as well as the position of cellular structures and orientation of cells within tissues. The centrosome serves as the main cytoskeleton-organizing centre in the cell and is the classical site of microtubule nucleation and anchoring. For these reasons, centrosomes play a very important role in morphogenesis, not just in the early stages of cell divisions but also in the later stages of organogenesis. Many organs such as lung, kidney and blood vessels develop from epithelial tubes that branch into complex networks. Cells in the nervous system also form highly branched structures in order to build complex neuronal networks. During branching morphogenesis, cells have to rearrange within tissues though multicellular branching or through subcellular branching, also known as single-cell branching. For highly branched structures to be formed during embryonic development, the cytoskeleton needs to be extensively remodelled. The centrosome has been shown to play an important role during these events.

The specification of tissue identity during embryonic development requires precise spatio-temporal coordination of gene expression. Many transcription factors required for the development of organs have been identified and their expression patterns are known; however, the mechanisms through which they coordinate gene expression in time remain poorly understood. Here, we show that hormone-induced transcription factor Blimp-1 participates in the temporal coordination of tubulogenesis in Drosophila melanogaster by regulating the expression of many genes involved in tube maturation. In particular, we demonstrate that Blimp-1 regulates the expression of genes involved in chitin deposition and F-actin organization. We show that Blimp-1 is involved in the temporal control of lumen maturation by regulating the beginning of chitin deposition. We also report that Blimp-1 represses a variety of genes involved in tracheal maturation. Finally, we reveal that the kinase Btk29A serves as a link between Blimp-1 transcriptional repression and apical extracellular matrix organization.

The morphology of organs, and hence their proper physiology, relies to a considerable extent on the extracellular matrix (ECM) secreted by their cells. The ECM is a structure contributed to and commonly shared by many cells in an organism that plays an active role in morphogenesis. Increasing evidence indicates that the ECM not only provides a passive contribution to organ shape but also impinges on cell behaviour and genetic programmes. The ECM is emerging as a direct modulator of many aspects of cell biology, rather than as a mere physical network that supports cells. Here, we review how the apical chitinous ECM is generated in Drosophila trachea and how cells participate in the formation of this supracellular structure. We discuss recent findings on the molecular and cellular events that lead to the formation of this apical ECM (aECM) and how it is influenced and affects tracheal cell biology.

© 2015 by the author; licensee MDPI, Basel, Switzerland. Cell migration and guidance are complex processes required for morphogenesis, the formation of tumor metastases, and the progression of human cancer. During migration, guidance molecules induce cell directionality and movement through complex intracellular mechanisms. Expression of these molecules has to be tightly regulated and their signals properly interpreted by the receiving cells so as to ensure correct navigation. This molecular control is fundamental for both normal morphogenesis and human disease. The Hedgehog (Hh) signaling pathway is evolutionarily conserved and known to be crucial for normal cellular growth and differentiation throughout the animal kingdom. The relevance of Hh signaling for human disease is emphasized by its activation in many cancers. Here, I review the current knowledge regarding the involvement of the Hh pathway in cell migration and guidance during Drosophila development and discuss its implications for human cancer origin and progression., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

© 2015 Elsevier Ireland Ltd. We report that the morphogen Hedgehog (Hh) is an axonal chemoattractant in the midline of Drosophila melanogaster embryos. Hh is present in the ventral nerve cord during axonal guidance and overexpression of hh in the midline causes ectopic midline crossing of FasIIpositive axonal tracts. In addition, we show that Hh influences axonal guidance via a noncanonical signalling pathway dependent on Ptc. Our results reveal that the Hh pathway cooperates with the Netrin/Frazzled pathway to guide axons through the midline in invertebrates., E.B. was supported by a fellowship from the Spanish Ministerio de Ciencia y Innovación, D.R. was supported by the Eramus programme and a FPU fellowship (FPU 12/05765) and S.J.A. acknowledges a Ramon y Cajal Researcher position granted by the Spanish Ministerio de Ciencia y Innovación. This work was funded by the Spanish Ministerio de Ciencia y Innovación (RYC-2007-00417)

The extracellular matrix (ECM), a structure contributed to and commonly shared by many cells in an organism, plays an active role during morphogenesis. Here, we used the Drosophila tracheal system to study the complex relationship between the ECM and epithelial cells during development. We show that there is an active feedback mechanism between the apical ECM (aECM) and the apical F-actin in tracheal cells. Furthermore, we reveal that cell-cell junctions are key players in this aECM patterning and organisation and that individual cells contribute autonomously to their aECM. Strikingly, changes in the aECM influence the levels of phosphorylated Src42A (pSrc) at cell junctions. Therefore, we propose that Src42A phosphorylation levels provide a link for the ECM environment to ensure proper cytoskeletal organisation. DOI: http://dx.doi.org/10.7554/eLife.09373.001, eLife digest Animal cells can secrete proteins and molecules into the space around them to create a support they can attach to. This structure – known as the extracellular matrix – comes in various forms and can help to shape tissues or influence the way in which cells behave. Inside cells, filaments made of a protein called actin also provide structural support. In fruit fly larvae, “tracheal” cells create a network of tubes that will form the airways of the adult fly. Once this network is complete, these cells secrete the materials to make an extracellular matrix in the internal (apical) surface of the tubes. This matrix has a series of spiralling ridges made from a molecule called chitin. These ridges run along the tubes, spanning several cells and providing the mechanical strength needed to keep the airways open. The ridges appear to form through a co-ordinated effort between the cells, and recent studies suggest that actin filaments may be involved in this process. Here, Öztürk-Çolak et al. investigate this idea further by carrying out a detailed analysis of the relationship between the extracellular matrix and the tracheal cells as the airways develop. The experiments reveal that rings of actin filaments form on the apical side of tracheal cells before the ridges appear. These rings generate regular folds in the membrane that surrounds each tracheal cell and are required for an enzyme to accumulate in the cells. This enzyme produces chitin, leading to its deposition in stripes above the actin rings. Further experiments show that the junctions between cells play an important role in organising the pattern of the extracellular matrix. The active form of a protein called Src42A – which is known to regulate the way actin filaments are organized inside cells – accumulates at these junctions. Excessive Src42A activity in tracheal cells alters the networks of actin filaments and disrupts the formation of the matrix. Öztürk-Çolak et al. also find evidence of a “feedback” mechanism, in which the presence of chitin reduces the activity of Src42A to maintain the correct patterning of actin. These findings reveal that actin and junctions between cells play a central role in co-ordinating the formation of the extracellular matrix in fruit fly airways. The next challenge will be to understand which proteins and other molecules are involved in the process that allows the extracellular matrix to communicate with the cells. DOI: http://dx.doi.org/10.7554/eLife.09373.002

Centrosome amplification is a hallmark of cancer, although we are still far from understanding how this process affects tumorigenesis [1, 2]. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. Here, we exploit the effects of centrosome amplification in post-mitotic cells during single-cell branching. We show that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. We found that mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen. We show that Rca1 and CycA post-mitotic cells have supernumerary centrosomes and that other mutant conditions that increase centrosome number also show excess of subcellular lumen branching. Furthermore, we show that de novo lumen formation is impaired in mutant embryos with fewer centrioles. The data presented here define a requirement for the centrosome as a microtubule-organizing center (MTOC) for the initiation of subcellular lumen formation. We propose that centrosomes are necessary to drive subcellular lumen formation. In addition, centrosome amplification increases single-cell branching, a process parallel to capillary sprouting in blood vessels [3]. These results shed new light on how centrosomes can contribute to pathology independently of mitotic defects., S.J.A. is a Ramon y Cajal Researcher (RYC-2007-00417); D.R. is the recipient of an FPU PhD fellowship from the Spanish Ministerio de Educación (FPU12/O5765); M.D. was supported by the Erasmus Programme. This work was supported by the Generalitat de Catalunya and grants from the Spanish Ministerio de Ciencia e Innovación/Ministerio de Economia y Competitividad (BFU2009-07629 and BFU2012-32115). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Spanish Government).

Assembly, maintenance and repair of nervous systems rely on the precise coordination in the presentation of guidance signals and the correct reception and processing of these signals. During embryonic development, considerable progress has already been made in identifying the extracellular cues and the receptors mediating axonal guidance (Araujo and Tear, 2003). Axons are particularly vulnerable to injury and disease and axonal damage plays a central role in neurodegenerative disorders. Hence, full integration of axonal guidance information will help us understand how cells can combine extensive extracellular information to follow an unerring migration pathway. In addition, this understanding will yield clues on how to encourage axonal regeneration after injury or disease.

Tramtrack (Ttk) is a widely expressed transcription factor, the function of which has been analysed in different adult and embryonic tissues in Drosophila. So far, the described roles of Ttk have been mainly related to cell fate specification, cell proliferation and cell cycle regulation. Using the tracheal system of Drosophila as a morphogenetic model, we have undertaken a detailed analysis of Ttk function. Ttk is autonomously and non-autonomously required during embryonic tracheal formation. Remarkably, besides a role in the specification of different tracheal cell identities, we have found that Ttk is directly involved and required for different cellular responses and morphogenetic events. In particular, Ttk appears to be a new positive regulator of tracheal cell intercalation. Analysis of this process in ttk mutants has unveiled cell shape changes as a key requirement for intercalation and has identified Ttk as a novel regulator of its progression. Moreover, we define Ttk as the first identified regulator of intracellular lumen formation and show that it is autonomously involved in the control of tracheal tube size by regulating septate junction activity and cuticle formation. In summary, the involvement of Ttk in different steps of tube morphogenesis identifies it as a key player in tracheal development.

Cell migration is a widespread and complex process that is crucial for morphogenesis and for the underlying invasion and metastasis of human cancers. During migration, cells are steered toward target sites by guidance molecules that induce cell direction and movement through complex intracellular mechanisms. The spatio-temporal regulation of the expression of these guidance molecules is of extreme importance for both normal morphogenesis and human disease. One way to achieve this precise regulation is by combinatorial inputs of different transcription factors. Here we used Drosophila melanogaster mutants with migration defects in the ganglionic branches of the tracheal system to further clarify guidance regulation during cell migration. By studying the cellular consequences of overactivated Hh signalling, using ptc mutants, we found that Hh positively regulates Bnl/FGF levels during embryonic stages. Our results show that Hh modulates cell migration non-autonomously in the tissues surrounding the action of its activity. We further demonstrate that the Hh signalling pathway regulates bnl expression via Stripe (Sr), a zinc-finger transcription factor with homology to the Early Growth Response (EGR) family of vertebrate transcription factors. We propose that Hh modulates embryonic cell migration by participating in the spatio-temporal regulation of bnl expression in a permissive mode. By doing so, we provide a molecular link between the activation of Hh signalling and increased chemotactic responses during cell migration. © 2014 Butí et al., This work was funded by the Spanish Ministerio de Ciencia y Innovación (RYC-2007-00417, BFU-2006-01935, BFU-2009-07629). E.B. was supported by a FPI fellowship and D.M. by a Leonardo da Vinci fellowship. S.J.A. holds a Ramon y Cajal Researcher position granted by the Spanish Ministerio de Ciencia y Innovación

In mammalian cells, the core factors involved in the damage recognition and incision steps of DNA nucleotide excision repair are XPA, TFIIH complex, XPC-HR23B, replication protein A (RPA), XPG, and ERCC1-XPF. Many interactions between these components have been detected, using different physical methods, in human cells and for the homologous factors in Saccharomyces cerevisiae. Several human nucleotide excision repair (NER) complexes, including a high-molecular-mass repairosome complex, have been proposed. However, there have been no measurements of activity of any mammalian NER protein complex isolated under native conditions. In order to assess relative strengths of interactions between NER factors, we captured TFIIH from cell extracts with an anti-cdk7 antibody, retaining TFIIH in active form attached to magnetic beads. Coimmunoprecipitation of other NER proteins was then monitored functionally in a reconstituted repair system with purified proteins. We found that all detectable TFIIH in gently prepared human cell extracts was present in the intact nine-subunit form. There was no evidence for a repair complex that contained all of the NER components. At low ionic strength TFIIH could associate with functional amounts of each NER factor except RPA. At physiological ionic strength, TFIIH associated with significant amounts of XPC-HR23B and XPG but not other repair factors. The strongest interaction was between TFIIH and XPC-HR23B, indicating a coupled role of these proteins in early steps of repair. A panel of antibodies was used to estimate that there are on the order of 10(5) molecules of each core NER factor per HeLa cell.

Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. Defects in NER result in three different human disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). Two cases with the combined features of XP and CS have been assigned to the XP–D complementation group. Despite their extreme UV sensitivity, these cells appeared to incise their DNA as efficiently as normal cells in response to UV damage. These incisions were, however, uncoupled from the rest of the repair process. Using cell-free extracts, we were unable to detect any incision activity in the neighbourhood of the damage. When irradiated plasmids were introduced into unirradiated XP–D/CS cells, the ectopically introduced damage triggered the induction of breaks in the undamaged genomic DNA. XP–D/CS cells thus have a unique response to sensing UV damage, which results in the introduction of breaks into the DNA at sites distant from the damage. We propose that it is these spurious breaks that are responsible for the extreme UV sensitivity of these cells.

During human nucleotide excision repair, damage is recognized, two incisions are made flanking a DNA lesion, and residues are replaced by repair synthesis. A set of proteins required for repair of most lesions is RPA, XPA, TFIIH, XPC–hHR23B, XPG, and ERCC1–XPF, but additional components have not been excluded. The most complex and difficult to analyze factor is TFIIH, which has a 6-subunit core (XPB, XPD, p44, p34, p52, p62) and a 3-subunit kinase (CAK). TFIIH has roles both in basal transcription initiation and in DNA repair, and several inherited human disorders are associated with mutations in TFIIH subunits. To identify the forms of TFIIH that can function in repair, recombinant XPA, RPA, XPC–hHR23B, XPG, and ERCC1–XPF were combined with TFIIH fractions purified from HeLa cells. Repair activity coeluted with the peak of TFIIH and with transcription activity. TFIIH from cells with XPB or XPD mutations was defective in supporting repair, whereas TFIIH from spinal muscular atrophy cells with a deletion of one p44 gene was active. Recombinant TFIIH also functioned in repair, both a 6- and a 9-subunit form containing CAK. The CAK kinase inhibitor H-8 improved repair efficiency, indicating that CAK can negatively regulate NER by phosphorylation. The 15 recombinant polypeptides define the minimal set of proteins required for dual incision of DNA containing a cisplatin adduct. Complete repair was achieved by including highly purified human DNA polymerase δ or ε, PCNA, RFC, and DNA ligase I in reaction mixtures, reconstituting adduct repair for the first time with recombinant incision factors and human replication proteins.

TFIIH is a multisubunit protein complex involved in RNA polymerase II transcription and nucleotide excision repair, which removes a wide variety of DNA lesions including UV-induced photoproducts. Mutations in the DNA-dependent ATPase/helicase subunits of TFIIH, XPB and XPD, are associated with three inherited syndromes as follows: xeroderma pigmentosum with or without Cockayne syndrome and trichothiodystrophy. By using epitope-tagged XPD we purified mammalian TFIIH carrying a wild type or an active-site mutant XPD subunit. Contrary to XPB, XPD helicase activity was dispensable for in vitro transcription, catalytic formation of trinucleotide transcripts, and promoter opening. Moreover, in contrast to XPB, microinjection of mutant XPD cDNA did not interfere with in vivo transcription. These data show directly that XPD activity is not required for transcription. However, during DNA repair, neither 5' nor 3' incisions in defined positions around a DNA adduct were detected in the presence of TFIIH containing inactive XPD, although substantial damage-dependent DNA synthesis was induced by the presence of mutant XPD both in cells and cell extracts. The aberrant damage-dependent DNA synthesis caused by the mutant XPD does not lead to effective repair, consistent with the discrepancy between repair synthesis and survival in cells from a number of XP-D patients.

Competition and determination of leading and trailing cells during collective cell migration is a widespread phenomenon in development, wound healing and tumour invasion. Here, we analyse this issue during in vivo ganglionic branch cell migration in the Drosophila tracheal system. We identify Sequoia (Seq) as a negative transcriptional regulator of Branchless (Bnl), a Drosophila FGF homologue, and observe that modulation of Bnl levels determines how many cells will lead this migrating cluster, regardless of Notch lateral inhibition. Our results show that becoming a tip cell does not prevent others in the branch taking the same position, suggesting that leader choice does not depend only on sensing relative amounts of FGF receptor activity. © 2011. Published by The Company of Biologists Ltd., S.J.A. is a Ramon y Cajal Researcher previously supported by an I3P postdoctoral contract from the Consejo Superior de Investigaciones Cientificas (CSIC) and a Beatriu de Pinós fellowship (AGAUR). This work is supported by grants from the Generalitat de Catalunya and the Spanish Ministerio de Ciencia e Innovación (BFU2009-07629 and Consolider CSD2007-00008).

Vertebrates and invertebrates share the formidable task of accurately establishing the elaborate connections that make up their nervous systems. Researchers investigating this process have the challenge of identifying the molecules and mechanisms that underlie this process. Each group of organisms offers their own advantages for piecing together the conserved constituents. Broadly speaking, the invertebrates have allowed the discovery of relevant genes through classical genetic screens for mutations that affect the process of axon guidance, whereas vertebrates provide numerous systems for the elaboration of the functional mechanisms. Here, we focus on the role of invertebrates in characterizing the molecular mechanisms of axon guidance.

Axonal growth and guidance rely on correct growth cone responses to guidance cues. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the crosstalk mechanisms between guidance and membrane dynamics and turnover. Recent studies indicate that whereas axonal attraction requires exocytosis, chemorepulsion relies on endocytosis. Indeed, our own studies have shown that Netrin-1/Deleted in Colorectal Cancer (DCC) signaling triggers exocytosis through the SNARE Syntaxin-1 (STX1). However, limited in vivo evidence is available about the role of SNARE proteins in axonal guidance. To address this issue, here we systematically deleted SNARE genes in three species. We show that loss-of-function of STX1 results in pre- and post-commissural axonal guidance defects in the midline of fly, chick, and mouse embryos. Inactivation of VAMP2, Ti-VAMP, and SNAP25 led to additional abnormalities in axonal guidance. We also confirmed that STX1 loss-of-function results in reduced sensitivity of commissural axons to Slit-2 and Netrin-1. Finally, genetic interaction studies in Drosophila show that STX1 interacts with both the Netrin-1/DCC and Robo/Slit pathways. Our data provide evidence of an evolutionarily conserved role of STX1 and SNARE proteins in midline axonal guidance in vivo, by regulating both pre- and post-commissural guidance mechanisms, This work was funded by MINECO (to ES: SAF2013-42445-R, SAF2016-7426 and BFU2010-21507; FU: RYC-2007-00417; and SJA: RYC-2009-05510), ISCIII (to ES: CIBERNED), Ministerio de Educación, Cultura y Arte (to OR: AP2005-1662), Fundació La Caixa (to PJB) and the Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (to ETS: 3100A_130730 and 31003A_166479).